Abstract
Objective
To evaluate IV indocyanine green (ICG) near-infrared fluorescence (NIRF) imaging to identify normal canine parathyroid tissue.
Methods
A cumulative effect study followed by a dose evaluation study with 8 purpose-bred dogs was performed from February through April 2023. Dogs were randomized to receive IV ICG at 0.2, 0.3, or 0.4 mg/kg after the thyroid and parathyroid glands were exposed. A NIRF endoscope positioned 8 cm above the thyroid-parathyroid complex obtained images. Subjective and objective measures of fluorescence were recorded and compared for the thyroid gland, external parathyroid gland, and internal parathyroid gland.
Results
Repeated ICG administration did not affect time to peak fluorescence but increased peak parathyroid gland fluorescence. Subjective fluorescence scores of the parathyroid glands were significantly higher in monochromatic modality compared to other ICG-NIRF modalities. Initial fluorescence was immediate for all glands. Mean time to peak objective fluorescence was 0.2 to 1.9 minutes. Higher ICG doses generally had higher peak fluorescence than lower ICG doses. Indocyanine green–NIRF did not consistently distinguish normal parathyroid glands from thyroid tissue.
Conclusions
ICG-NIRF at 0.2 to 0.4 mg/kg effectively fluoresces normal parathyroid glands in dogs, although the subjective fluorescence achieved in the parathyroid glands is similar to fluorescence in the adjacent thyroid glands. Parathyroid fluorescence varied substantially between ICG-NIRF modality, with the highest fluorescence observed in the monochromatic modality.
Clinical Relevance
ICG-NIRF may aid in intraoperative localization of parathyroid glands, particularly the identification of ectopic parathyroid tissue. Further evaluation of ICG-NIRF for the identification of pathologic parathyroid tissue in clinical patients is indicated.
The identification and removal of abnormal parathyroid glands is critical for successful treatment of primary hyperparathyroidism in dogs. Because size and positioning of parathyroid glands vary among dogs and abnormal parathyroid glands may be similar in size to normal glands, the identification of abnormal parathyroid glands in dogs with primary hyperparathyroidism presents a clinical challenge.1–3 Hypocalcemia is a common postoperative complication that can be exacerbated by unnecessary parathyroid tissue removal.2,4–6 The need for postoperative calcium supplementation can prolong hospitalization and increase costs to the client.7 Conversely, failure to remove abnormal parathyroid tissue may result in persistent hypercalcemia and additional surgery and morbidity.8,9
Cervical ultrasound to identify abnormal parathyroid glands preoperatively has limited sensitivity, and surgical findings may differ from preoperative ultrasound findings.10,11 Intraoperative parathyroid hormone measurements may be utilized after surgical excision of the suspected pathologic gland to confirm successful removal of hyperfunctional tissue; however, rapid parathyroid hormone assays are not widely available, and postresection samples are typically obtained 10 to 20 minutes following excision, leading to increased surgical and anesthesia time.12 Intravenous methylene blue administration has also been evaluated for intraoperative differentiation of abnormal parathyroid tissue but showed limited efficacy and may be associated with Heinz body anemia and acute renal failure.13 An accurate and safe real-time method for intraoperative identification of pathological and ectopic parathyroid tissue could help improve the treatment of dogs with primary hyperparathyroidism.
Near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) has recently emerged as a real-time intraoperative diagnostic to identify parathyroid glands in humans.14,15 Indocyanine green has a well-established safety profile, but effective dosage varies greatly between species and procedures.16 In dogs, reported doses range from 0.02 to 5.0 mg/kg depending on the procedure and route of ICG administration. Based on a single study,17 ICG and NIRF imaging were successful in visualizing fluorescence of the external parathyroid glands in 3 dogs at an optimal ICG dose of 0.018 mg/kg. This study did not report fluorescence of the internal parathyroid glands nor document the time between repeated ICG injections and whether repeated ICG administration affected parathyroid gland fluorescence. Furthermore, newer NIRF imaging platforms provide additional modalities for viewing NIRF, including a traditional monochromatic modality as well as overlay and intensity map modalities that allow the surgeon to view ICG fluorescence overlaid with white light illumination of tissue and with colorized representation of fluorescence intensity, respectively.
The objectives of this study were to (1) evaluate for a cumulative dose effect on the fluorescence of parathyroid glands with repeated ICG administration, (2) measure the time to initial and peak parathyroid fluorescence following IV ICG administration, and (3) compare external parathyroid, internal parathyroid, and thyroid fluorescence at multiple ICG doses across ICG-NIRF modalities to evaluate optimal IV ICG dosing for differentiating normal parathyroid tissue.
Methods
Patient selection
Eight purpose-bred dogs were enrolled in this study. A cumulative effect study was performed with 2 dogs to refine ICG dosing and to evaluate for a cumulative effect on parathyroid fluorescence with repeated ICG administration. Subsequently, 6 dogs were included in the dose evaluation study. Approval was obtained by the IACUC (protocol ID No. 22-366), and the study was performed from February through April of 2023.
Procedure
A board-certified surgeon performed all surgeries with the assistance of a surgical resident. An IV catheter was placed in the right cephalic vein for the facilitation of anesthetic agents and ICG administration. All dogs were anesthetized with a standardized protocol and placed in dorsal recumbency. A ventral midline cervical approach was made extending from the caudal aspect of the larynx to approximately 4 cm cranial to the manubrium. A combination of blunt and sharp dissection was used to expose and evaluate the left and right thyroid-parathyroid complexes. Weitlaner retractors (V. Mueller) were placed to maintain exposure. Size, shape, location, visibility, and any abnormalities of all external and internal parathyroid glands were documented prior to ICG administration. The operating room was darkened by turning off all room lights and covering windows prior to ICG administration. Following data collection, the surgical site was closed routinely, and all dogs recovered from anesthesia with postoperative analgesia and monitoring. At the conclusion of the study, the dogs were made available for adoption as approved by the IACUC.
Indocyanine green dosing
Indocyanine green contrast solution was prepared by reconstituting a 25-mg vial of ICG (Pantheon) with 10 mL of sterile water. Based on the ICG dose to detect canine external parathyroid glands in a previous study,17 a dose of 0.015 mg/kg ICG was initially selected for cumulative effect study dog 1. No fluorescence of the thyroid or parathyroid glands was appreciated after IV administration of 0.015 mg/kg ICG. The dose was subsequently increased to 0.2 mg/kg for the first dog and 0.4 mg/kg for the second dog. Cumulative effect study dog 1 was administered 0.2 mg/kg ICG every 20 minutes for a total of 5 doses, with each dose separated by a 20-minute washout period. Cumulative effect study dog 2 was administered 0.4 mg/kg ICG every 20 minutes for a total of 5 doses, with each dose separated by a 20-minute washout period.
Using a randomized block design, dogs for the dose evaluation study received ICG doses of 0.2, 0.3, or 0.4 mg/kg. Specifically, dose evaluation study dogs 2 and 3 were administered 2 doses of 0.2 mg/kg ICG separated by a 30-minute washout period. Dose evaluation study dogs 1 and 4 were administered 2 doses of 0.3 mg/kg ICG separated by a 30-minute washout period, and dogs 5 and 6 were administered 2 doses of 0.4 mg/kg ICG separated by a 30-minute washout period.
Data collection
A 30°, 5-mm endoscope (Rubina; Karl Storz) was positioned 8 cm above the thyroid-parathyroid complex and held in place with a VITOM (Karl Storz) for image capture of the left and right thyroid and parathyroid glands. Once all thyroid and parathyroid glands visible under white light were initially identified, the most readily visible internal parathyroid gland, external parathyroid gland, and thyroid gland were chosen for positioning in the field of view for image capture to allow simultaneous observation of fluorescence for all 3 glands. For the cumulative effect study, images were captured every 60 seconds from the time of ICG administration until 20 minutes after ICG administration. For the dose evaluation study, images were captured every 120 seconds from the time of ICG administration until 30 minutes after ICG administration.
At each time point during the procedure, subjective scores of fluorescence for the thyroid gland, external parathyroid gland, and internal parathyroid gland in monochromatic, overlay, and intensity map modality were recorded by the unblinded primary investigator using a 5-point scale developed by the investigators for this study (Supplementary Table S1). Post hoc images captured in monochromatic mode were later analyzed using image software (ImageJ, version 1.54h; NIH) to quantify the degree of external and internal parathyroid and thyroid gland fluorescence at each time point. Objective fluorescence was reported in counts per pixel in arbitrary units.
Statistical analysis
Descriptive analyses of qualitative and quantitative measures of fluorescence at each time point were reported. All results for objective fluorescence were analyzed and reported for monochromatic modality only; all results for subjective fluorescence were analyzed and reported for monochromatic, overlay, and intensity map modalities. Graphical methods, including normal quantile-quantile plots and histograms, were used to assess the normality of continuous variables. Normally distributed data were expressed as means with SD, and non-normally distributed data were expressed as medians with IQR. Spearman correlations were calculated to determine cumulative dose effects. Both linear mixed-effects models (R lme4) and ordinal logistic mixed-effects models (R ordinal) were utilized to evaluate fluorescence based on dose, gland, and time.18,19 Post hoc pairwise comparisons (with Tukey adjustments for multiple testing where appliable) were used to compare fluorescence at different time points across modalities and doses. Statistical analysis was performed by a statistician using R statistical software, version 4.4.0 (R Foundation for Statistical Computing). P < .05 indicated statistical significance.
Results
Sample population
All dogs were purpose-bred neutered hounds. The median age was 3.8 years (range, 2.9 to 3.9 years). The median weight was 24.1 kg (range, 23.3 to 27.8 kg). No visible pathology was observed in the thyroid or parathyroid glands of any dog. In 4 out of 8 dogs, the thyroid gland, external parathyroid glands, and internal parathyroid glands were able to be identified bilaterally. The right internal parathyroid gland could not be visualized in 2 dogs, and the left internal parathyroid gland could not be visualized in 2 dogs. Glands utilized for subjective and objective fluorescence evaluation were chosen based on the ability to simultaneously visualize the thyroid gland, external parathyroid gland, and internal parathyroid gland without additional manipulation following ICG administration; the observed glands in each dog are listed in Supplementary Table S2.
Indocyanine green dosing
Cumulative effect study dog 1 was initially administered a dose of 0.015 mg/kg ICG; no fluorescence of any tissues was observed during the 20 minutes following ICG administration. Cumulative effect study dog 1 was subsequently administered 0.2 mg/kg ICG, and cumulative effect study dog 2 was administered 0.4 mg/kg ICG. Indocyanine green doses of 0.2, 0.3, or 0.4 mg/kg were chosen for the dose evaluation study.
Part 1: cumulative effect study
Dose accumulation effect—There was no difference in time to peak fluorescence of the parathyroid glands with repeated ICG doses (P = .089 to P = .99). Peak fluorescence, however, showed a strong positive correlation with repeated ICG dose administration (ρ = 1; P = .017) with the exception of the external parathyroid gland at the 0.4-mg/kg dose (ρ = 0.9; P = .083). Repeated ICG administration increased the intensity of subsequent parathyroid and thyroid gland fluorescence (Figure 1).
Dose accumulation effect of thyroid and parathyroid gland fluorescence with repeated indocyanine green (ICG) administration over 180 minutes in 2 dogs. The y-axis represents peak objective fluorescence reported in counts per pixel in arbitrary units. The x-axis represents repeated ICG dose administration for dog 1 (0.2 mg/kg ICG X 5 doses) and dog 2 (0.4 mg/kg ICG X 5 doses) in the cumulative effect study. Data were collected every 60 seconds for 20 minutes for 5 doses. Post hoc images captured in monochromatic mode were analyzed using ImageJ to evaluate objective fluorescence. EPG = External parathyroid gland. IPG = Internal parathyroid gland. TG = Thyroid gland.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0371
Fluorescence by modality—At the 0.2-mg/kg dose, subjective fluorescence scores of the external and internal parathyroid glands were significantly higher in monochromatic modality compared to intensity map and overlay modalities at 1, 10, and 20 minutes following ICG administration (Figure 2). Subjective fluorescence scores in intensity map modality were higher than those with overlay at 1 minute (P = .026 and P = .007 for external and internal, respectively) but lower at 20 minutes (P = .001) and not significantly different at 10 minutes (P = .523 and P = .506 for external and internal, respectively). These findings were consistent for the 0.4-mg/kg dose with the exception that monochromatic scores were not significantly higher than overlay scores at 1 minute post ICG administration.
For the cumulative effect study, as described in Figure 1, subjective fluorescence scores were higher in monochromatic modality (A) compared to overlay modality (B) when 0.2 mg/kg of ICG was administered. Subjective fluorescence was measured using a 5-point scale. The white arrow indicates the external parathyroid gland. The white arrowhead indicates the internal parathyroid gland.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0371
Part 2: dose evaluation study
Initial fluorescence by dose and modality—Initial fluorescence was rapid and appeared by the time initial observation images were captured at time 0 for all glands. On post hoc pairwise comparison, objective measures of initial fluorescence of the external parathyroid gland for the 0.3- and 0.4-mg/kg doses were significantly greater than for the 0.2-mg/kg dose (P = .025 and P = .005, respectively). Similarly, the initial objective fluorescence of the thyroid gland was significantly greater with the 0.4-mg/kg dose compared to the 0.2-mg/kg dose (P = .010). No significant differences were found between ICG dose and initial fluorescence of the internal parathyroid gland.
No difference between subjective measures of initial fluorescence was detected in monochromatic modality for any gland. In both the intensity map and overlay modalities, the external parathyroid gland had greater odds of a high fluorescence score with the 0.3- and 0.4-mg/kg doses compared to the 0.2-mg/kg dose (P = .037 and P = .013, respectively, for intensity map; P = .013 and P = .013, respectively, for overlay). The thyroid gland had greater odds of a high fluorescence score at the 0.4-mg/kg dose compared to the 0.2-mg/kg dose in the intensity map modality (P = .031).
Initial parathyroid gland fluorescence compared to thyroid gland fluorescence
The thyroid and external parathyroid gland had significantly greater mean initial fluorescence than the internal parathyroid gland at the 0.4-mg/kg dose only (P = .005 and P = .014, respectively); no differences were detected in initial objective fluorescence between glands at the 0.2- and 0.3-mg/kg doses. In both the intensity map and overlay modalities, the external parathyroid was more likely to have a high fluorescence score than the internal parathyroid and thyroid gland at the 0.2-mg/kg dose (Table 1). In the intensity map modality at the 0.4-mg/kg dose, the thyroid gland was less likely to have a high fluorescence score than the external and internal parathyroid glands (P = .045 and P = .002, respectively). In overlay modality, the internal parathyroid gland was more likely to have a high fluorescence score than the thyroid gland at the 0.4-mg/kg dose (P = .020).
Summary of post hoc pairwise comparisons for initial subjective fluorescence of the parathyroid glands and thyroid gland in intensity map and overlay modality for dogs in the dose evaluation study.
| Modality | Dose (mg/kg) | Glands | OR | 95% CI | χ2 | P value |
|---|---|---|---|---|---|---|
| Intensity map | 0.2 | EPG vs IPG | 10,366.09 | 4.69–22,892,145.05 | 7.92 | .014* |
| EPG vs TG | 10,366.16 | 4.69–22,890,210.47 | 7.92 | .014* | ||
| IPG vs TG | 1.00 | 0.01–91.76 | 0.00 | > .99 | ||
| 0.4 | EPG vs IPG | 0.07 | 0.00–4.09 | 2.33 | .278 | |
| EPG vs TG | 144.02 | 1.09–18,977.67 | 5.69 | .045* | ||
| IPG vs TG | 1,996.36 | 10.58–376,541.99 | 11.55 | .002* | ||
| Overlay | 0.2 | EPG vs IPG | 10,366.16 | 4.69–22,893,879.67 | 7.92 | .014* |
| EPG vs TG | 10,366.27 | 4.69–22,894,091.92 | 7.92 | .014* | ||
| IPG vs TG | 1.00 | 0.01–91.76 | 0.00 | > .99 | ||
| 0.4 | EPG vs IPG | 0.08 | 0.00–5.71 | 1.89 | .354 | |
| EPG vs TG | 19.71 | 0.27–1,465.50 | 2.63 | .236 | ||
| IPG vs TG | 233.99 | 2.02–27,119.25 | 7.24 | .020* |
*Values of P < .05 were considered statistically significant.
EPG = External parathyroid gland. IPG = Internal parathyroid gland. TG = Thyroid gland.
Subjective fluorescence was measured using a 5-point scale. The dose evaluation study was performed in 6 dogs, with each dog receiving 2 doses of the designated indocyanine green dose separated by a 30-minute washout period. Data were collected every 120 seconds for 30 minutes.
Objective peak fluorescence
Mean time to objective peak fluorescence of the parathyroid glands ranged from 0.2 to 1.9 minutes (Table 2). The internal parathyroid gland had a significantly longer time to peak objective fluorescence compared to the external parathyroid and thyroid (P = .001 and P = .004, respectively). Peak objective fluorescence of the parathyroid glands ranged from 42 to 135 counts/pixel in arbitrary units, with higher doses generally having a higher peak fluorescence. As shown (Figure 3), the 0.3- and 0.4-mg/kg doses resulted in greater objective peak fluorescence for the external parathyroid and thyroid gland than the 0.2-mg/kg dose (P = .001 and P < .001, respectively, for external parathyroid; P = .005 and P < .001, respectively, for thyroid); only the 0.4-mg/kg dose resulted in greater peak objective fluorescence of the internal parathyroid gland compared to the 0.2-mg/kg dose (P = .016). Objective peak fluorescence did not vary between glands with the exception of the internal parathyroid gland peak fluorescence measuring significantly lower than the external parathyroid and thyroid glands at 0.4 mg/kg (P = .004 and P = .001, respectively).
Mean time to peak objective fluorescence after administration of 0.2, 0.3, and 0.4 mg/kg of indocyanine green for the dose evaluation study as described in Table 1.
| Dose (mg/kg) | Gland | Mean time of peak fluorescence (min) | 95% CI |
|---|---|---|---|
| 0.2 | EPG | 0.455 | 0.063–3.282 |
| 0.2 | IPG | 0.891 | 0.122–6.500 |
| 0.2 | TG | 0.632 | 0.087–4.580 |
| 0.3 | EPG | 0.192 | 0.025–1.463 |
| 0.3 | IPG | 1.882 | 0.247–14.353 |
| 0.3 | TG | 0.462 | 0.064–3.346 |
| 0.4 | EPG | 0.330 | 0.045–2.414 |
| 0.4 | IPG | 1.160 | 0.147–9.167 |
| 0.4 | TG | 0.252 | 0.035–1.839 |
Post hoc images captured in monochromatic mode were analyzed using ImageJ to evaluate objective fluorescence.
Representative image capture of peak objective fluorescence after IV administration of 0.2 mg/kg (A), 0.3 mg/kg (B), and 0.4 mg/kg (C) of ICG during the dose evaluation study. Post hoc images captured in monochromatic mode were analyzed using ImageJ to evaluate objective fluorescence. The dose evaluation study was performed in 6 dogs, with each dog receiving 2 doses of the designated ICG dose separated by a 30-minute washout period. Data were collected every 120 seconds for 30 minutes. The white arrow indicates the external parathyroid gland.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0371
Subjective peak fluorescence
Subjective scores for peak fluorescence of all glands in the 3 modalities can be found in Supplementary Table S3. For subjective fluorescence of the external parathyroid gland, the 0.4-mg/kg dose had greater odds of a high fluorescence score than the 0.2-mg/kg dose in intensity map modality (P = .023), and both the 0.3- and 0.4-mg/kg doses had greater odds of a high fluorescence score than the 0.2-mg/kg dose in overlay modality (P = .048 and P = .045, respectively).
In overlay and intensity map modality at the 0.2-mg/kg dose, the external parathyroid gland had greater odds of a high fluorescence score than the thyroid gland (P = .002 and P = .001, respectively). In overlay and intensity map modality at the 0.4-mg/kg dose, the internal parathyroid gland had greater odds of a high fluorescence score than the thyroid gland (P = .011 and P = .008, respectively). No other significant differences were found between subjective peak fluorescence of the thyroid glands and parathyroid glands; in particular, there were no significant differences in peak gland fluorescence between the thyroid and parathyroid glands in the monochromatic modality at any dose (P = .242, P = .056, and P = .187 at the 0.2-, 0.3-, and 0.4-mg/kg doses, respectively).
Progression and persistence of fluorescence
When evaluating fluorescence of the 3 glands at the 0-, 15-, and 30-minute time points, both the external parathyroid gland and thyroid gland showed significantly greater mean objective fluorescence at the 0.3- and 0.4-mg/kg doses (Table 3). At the 0.4-mg/kg dose, mean objective fluorescence decreased significantly faster for the external parathyroid gland and thyroid gland than for the internal parathyroid gland (P = .002 and P = .003, respectively), and the mean subjective fluorescence similarly decreased faster for the external parathyroid gland than for the internal parathyroid gland (P = .010).
Summary of post hoc pairwise comparisons of fluorescence at 0-, 15-, and 30-minute intervals for each gland and dose as described in Table 1, revealing persistence of objective fluorescence throughout the dose evaluation study.
| Time (min) | Gland | Dose comparison (mg/kg) | Ratio of mean fluorescence between doses | 95% CI | χ2 | P value |
|---|---|---|---|---|---|---|
| 0 | EPG | 0.3 vs 0.2 | 2.397 | 1.254–4.585 | 9.99 | .004* |
| 0.4 vs 0.2 | 3.595 | 1.880–6.876 | 21.39 | < .001* | ||
| 0.4 vs 0.3 | 1.500 | 0.784–2.868 | 2.15 | .308 | ||
| IPG | 0.3 vs 0.2 | 1.666 | 0.871–3.187 | 3.40 | .155 | |
| 0.4 vs 0.2 | 2.017 | 1.054–3.857 | 6.43 | .030* | ||
| 0.4 vs 0.3 | 1.210 | 0.633–2.315 | 0.48 | .769 | ||
| TG | 0.3 vs 0.2 | 2.117 | 1.107–4.049 | 7.35 | .018* | |
| 0.4 vs 0.2 | 3.353 | 1.754–6.413 | 19.13 | < .001* | ||
| 0.4 vs 0.3 | 1.584 | 0.828–3.029 | 2.77 | .219 | ||
| 15 | EPG | 0.3 vs 0.2 | 2.481 | 1.309–4.701 | 11.10 | .002* |
| 0.4 vs 0.2 | 3.397 | 1.793–6.438 | 20.11 | < .001* | ||
| 0.4 vs 0.3 | 1.369 | 0.723–2.595 | 1.33 | .482 | ||
| IPG | 0.3 vs 0.2 | 1.830 | 0.966–3.469 | 4.91 | .068 | |
| 0.4 vs 0.2 | 2.233 | 1.178–4.231 | 8.68 | .009* | ||
| 0.4 vs 0.3 | 1.220 | 0.644–2.312 | 0.53 | .746 | ||
| TG | 0.3 vs 0.2 | 2.371 | 1.251–4.493 | 10.02 | .004* | |
| 0.4 vs 0.2 | 3.358 | 1.772–6.363 | 19.73 | < .001* | ||
| 0.4 vs 0.3 | 1.416 | 0.747–2.684 | 1.63 | .409 | ||
| 30 | EPG | 0.3 vs 0.2 | 2.567 | 1.342–4.910 | 11.61 | .002* |
| 0.4 vs 0.2 | 3.210 | 1.678–6.140 | 17.77 | < .001* | ||
| 0.4 vs 0.3 | 1.250 | 0.654–2.391 | 0.65 | .698 | ||
| IPG | 0.3 vs 0.2 | 2.011 | 1.051–3.846 | 6.37 | .031* | |
| 0.4 vs 0.2 | 2.472 | 1.293–4.728 | 10.70 | .003* | ||
| 0.4 vs 0.3 | 1.230 | 0.643–2.351 | 0.56 | .735 | ||
| TG | 0.3 vs 0.2 | 2.656 | 1.389–5.080 | 12.47 | .001* | |
| 0.4 vs 0.2 | 3.363 | 1.758–6.431 | 19.22 | < .001* | ||
| 0.4 vs 0.3 | 1.266 | 0.662–2.421 | 0.73 | .670 |
*Values of P < .05 were considered statistically significant.
Post hoc images captured in monochromatic mode were analyzed using ImageJ to evaluate objective fluorescence.
Final fluorescence of all glands was measured at 30 minutes following ICG administration. Final objective fluorescence of the external parathyroid and thyroid glands was higher with the 0.3- and 0.4-mg/kg doses than with the 0.2-mg/kg dose (P = .014 and P = .003, respectively, for external parathyroid; P = .016 and P = .002, respectively, for thyroid) and was higher with the 0.4-mg/kg dose than the 0.2-mg/kg dose for the internal parathyroid gland (P = .033). Persistence of objective fluorescence was recorded in all glands throughout the 30-minute observation, whereas subjective fluorescence of all glands was absent in the intensity map and overlay modalities by 30 minutes following ICG administration at the 0.2- and 0.3-mg/kg doses, and faint fluorescence (subjective fluorescence scores < 1) remained in some glands in monochromatic modality at the 0.2- and 0.3-mg/kg dose.
Discussion
This study demonstrated rapid and persistent fluorescence of normal external and internal parathyroid glands with NIRF imaging with IV administration of ICG at doses ranging from 0.2 to 0.4 mg/kg. Higher ICG doses generally had higher fluorescence, although there was substantial variation in fluorescence between dose, gland, and modality. While ICG-NIRF did not consistently distinguish normal parathyroid from thyroid tissue, the results from this study support further evaluation of ICG-NIRF imaging as a potential intraoperative method for localization of ectopic or pathologic parathyroid tissue since fluorescence of parathyroid tissue was attainable.
Numerous reports have demonstrated the benefits of NIRF imaging and ICG administration to enhance the intraoperative identification of parathyroid glands in humans.14,15,20–24 Suh et al17 was the first study to evaluate ICG-NIRF imaging for visualization of the external parathyroid glands in dogs. The ICG dose reported in the Suh et al17 study, however, did not result in fluorescence of any tissue in the first dog in our study. This difference in fluorescence may be attributable to differences in NIRF imaging platform, endoscope type and positioning relative to the parathyroid tissue, or a combination of these factors.25–27 The cumulative effect study ICG doses were consequently increased to 0.2 and 0.4 mg/kg, resulting in successful fluorescence of the thyroid-parathyroid complexes. This finding highlights the potential variation in tissue fluorescence across imaging platforms and with varying distance from the observed tissue. While we standardized the variables of endoscope positioning and chose to use a commonly utilized NIRF imaging platform, it is important to note that the applicability of our findings to other NIRF systems, particularly the peak fluorescence achieved with a given ICG dose, may be subject to these limitations.
Monochromatic imaging has historically been a commonly available modality for NIRF angiography.23,28,29 The fluorescence of parathyroid glands in this study was consistently higher in monochromatic modality compared to intensity map or overlay modality across all doses. This is clinically significant as it suggests that monochromatic modality may be more useful for the evaluation of ectopic parathyroid tissue. Since the evaluation of the overlay and intensity map modalities was only performed using the subjective scoring and not objective analysis, comparisons across modality are subject to the limitations inherent with subjective scoring. Subjective scores were recorded by the primary surgeon who was unblinded to the dose and modality. This may have led to bias influencing subjective scores, particularly since the surgeon was also aware of the location of the parathyroid glands in each dog prior to observing gland fluorescence. This limits extrapolation to predicting the utility of fluorescence of ectopic parathyroid tissue since the surgeon in that clinical scenario is more likely to be unaware of the specific location of ectopic tissue. Limitations of monochromatic modality include a reduced ability to dissect tissues while in monochromatic modality because of the absence of white light illuminating surrounding tissue to facilitate safe visualization and dissection. Overlay and intensity map modalities are intended to allow the surgeon to visualize both the fluorescent tissue and surrounding tissue to facilitate dissection. Because fluorescence of the parathyroid glands was lower in overlay and intensity map modalities, the utility of ICG-NIRF for tissue dissection may be limited, but the strong fluorescence in monochromatic modality showed promise for aiding in parathyroid tissue localization.
Initial fluorescence occurred almost immediately for all glands when 0.2 to 0.4 mg/kg ICG was administered. Peak fluorescence of the parathyroid glands was also rapid (0.2 to 1.9 minutes), indicating that the ICG-NIRF at the doses evaluated allow for timely intraoperative identification of the internal and external parathyroid glands. Although higher ICG doses generally had higher fluorescence, there was substantial variation between modalities and dose. With few exceptions, we did not observe a statistically significant difference in fluorescence between the external and internal parathyroid glands and thyroid glands. These findings suggest that ICG-NIRF is likely not useful for differentiating normal parathyroid tissue from thyroid tissue, although investigation of the use of ICG-NIRF to identify and differentiate abnormal parathyroid tissue from normal parathyroid and thyroid tissue is warranted. These findings also pose a challenge to recommending a specific dose among those evaluated in this study for identifying parathyroid tissue. Based on our findings, specific doses could be selected for intended purposes. For example, if explicitly evaluating the internal parathyroid gland in overlay and intensity map modality, the internal parathyroid gland had greater odds of a higher fluorescence score than the thyroid gland when 0.4 mg/kg ICG was administered. Further evaluation in clinical patients is needed to determine the clinical utility of ICG for identifying parathyroid pathology and to refine ICG dosing for parathyroid pathology.
Objective fluorescence of all glands persisted throughout the 30-minute observation period. Similarly, subjective final fluorescence of all glands was absent in the intensity map and overlay modalities by 30 minutes following ICG administration at the 0.2- and 0.3-mg/kg doses but remained present in monochromatic modality throughout the observation period. This persistence of fluorescence is clinically useful for allowing exploration of the neck and evaluation of potential parathyroid tissue during the period of fluorescence. Of note, because subjective fluorescence persisted longer in monochromatic modality, switching to monochromatic modality may help facilitate continued exploration for parathyroid tissue. We also found that repeated ICG administration increased parathyroid gland fluorescence. This is important to recognize in that repeating IV ICG administration intraoperatively could increase parathyroid fluorescence if fluorescence begins to fade during surgical evaluation and/or more fluorescence is desired.
The limitations of this study include a limited range of ICG doses chosen for evaluation based on previous literature and the cumulative effect study; evaluation of a wider range of doses could allow for refinement of an optimal dose recommendation for normal parathyroid visualization. Due to variation in fluorescence with endoscope type and positioning, the findings of this study, particularly peak fluorescence, may not accurately extrapolate to other endoscopes of different sizes and distances from the tissue as well as to other NIRF imaging platforms. Additionally, the use of a small sample size of healthy dogs without apparent parathyroid pathology did not allow for assessment of whether ICG-NIRF may differentiate abnormal and normal parathyroid tissue. Future studies in dogs with parathyroid pathology are needed to evaluate the clinical application of ICG administration for the identification of pathologic parathyroid tissue.
In summary, ICG-NIRF imaging resulted in fluorescence of normal external and internal parathyroid glands in dogs. Subjective evaluation of peak fluorescence varied extensively across ICG modality. Based on the results of this study, ICG-NIRF imaging at the reported doses shows promise for aiding in the intraoperative identification of parathyroid tissue. Distinguishing parathyroid from thyroid tissue with ICG-NIRF is challenging, and further clinical investigation of ICG-NIRF imaging for the identification of pathological parathyroid tissue is indicated.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors thank Sarah Lewis for assistance with equipment acquisition.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
Funding for this study was provided by the American College of Veterinary Surgeons Foundation Surgery Resident Research Grant and Veterinary Endoscopy Society Research Grant.
ORCID
M. L. Oblak https://orcid.org/0000-0001-8489-4643
References
- 1.↑
Liles SR, Linder KE, Cain B, Pease AP. Ultrasonography of histologically normal parathyroid glands and thyroid lobules in normocalcemic dogs. Vet Radiol Ultrasound. 2010;51(4):447–452. doi:10.1111/j.1740-8261.2010.01686.x
- 2.↑
Townsend KL, Ham KM. Current concepts in parathyroid/thyroid surgery. Vet Clin North Am Small Amin Pract. 2022;52(2):455–471. doi:10.1016/j.cvsm.2021.12.004
- 3.↑
Cordella A, Bertaccini J, Rondena M, Andrea Z, Bertolini G. Multidetector-row CT findings in dogs with different primary parathyroid gland diseases. Vet Sci. 2022;9(6):273.
- 4.↑
Reinhart JM, Nuth EK, Byers CG, et al. Pre-operative fibrous osteodystrophy and severe, refractory, post-operative hypocalcemia following parathyroidectomy in a dog. Can Vet J. 2015;56(8):867–871.
- 5.
Galvao J, Chew D. Metabolic complications of endocrine surgery in companion animals. Vet Clin North Am Small Amin Pract. 2011;41(5):847–863. doi:10.1016/j.cvsm.2011.05.012
- 6.↑
Milovancev M, Schmiedt CW. Preoperative factors associated with postoperative hypocalcemia in dogs with primary hyperparathyroidism that underwent parathyroidectomy: 62 cases (2004-2009). J Am Vet Med Assoc. 2013;242(4):507–515. doi:10.2460/javma.242.4.507
- 7.↑
Dear JD, Kass PH, Dellla Maggiore AM, Feldman EC. Association of hypercalcemia before treatment with hypocalcemia after treatment in dogs with primary hyperparathyroidism. J Vet Intern Med. 2017;31(2):349–354. doi:10.1111/jvim.14644
- 8.↑
Feldman EC, Hoar B, Pollard R, Nelson RW. Pretreatment clinical and laboratory findings in dogs with primary hyperparathyroidism: 210 cases (1987-2004). J Am Vet Med Assoc. 2005;227(5):756–761. doi:10.2460/javma.2005.227.756
- 9.↑
Gear RNA, Neiger R, Skelly BJS, Herrtage ME. Primary hyperparathyroidism in 29 dogs: diagnosis, treatment, outcome and associated renal failure. J Small Anim Pract. 2005;46(1):10–16. doi:10.1111/j.1748-5827.2005.tb00268.x
- 10.↑
Wisner ER, Penninck S, Biller DS, Feldman EC, Drake C, Nyland TG. High-resolution parathyroid sonography. Vet Radiol Ultrasound. 1997;38(6):462–466. doi:10.1111/j.1740-8261.1997.tb00872.x
- 11.↑
Burkhardt SJ, Sumner JP, Mann S. Ambidirectional cohort study on the agreement of ultrasonography and surgery in the identification of parathyroid pathology, and predictors of postoperative hypocalcemia in 47 dogs undergoing parathyroidectomy due to primary hyperparathyroidism. Vet Surg. 2021;50(7):1379–1388. doi:10.1111/vsu.13707
- 12.↑
Ham K, Greenfield CL, Barger A, et al. Validation of a rapid parathyroid hormone assay and intraoperative measurement of parathyroid hormone in dogs with benign naturally occurring primary hyperparathyroidism. Vet Surg. 2009;38(1):122–132. doi:10.1111/j.1532-950X.2008.00457.x
- 13.↑
Feldman EC. Hypocalcemia and primary hypoparathyroidism. In: Feldman EC, Nelson RW, Reusch CE, Scott-Moncrieff JCR, eds. Canine and Feline Endocrinology. 4th ed. Elsevier Saunders; 2015:625–648.
- 14.↑
Matson J, Lwin TM, Bouvet M. Rapid intraoperative perfusion assessment of parathyroid adenomas with ICG using a wide-field portable hand-held fluorescence imaging system. Am J Surg. 2022;223(4):686–693. doi:10.1016/j.amjsurg.2021.07.027
- 15.↑
Zaidi N, Bucak E, Okoh A, Yazici P, Tigitbas H, Berber E. The utility of indocyanine green near infrared fluorescent imaging in the identification of parathyroid glands during surgery for primary hyperparathyroidism. J Surg Onc. 2016;113(7):771–774. doi:10.1002/jso.24240
- 16.↑
Cherrick GR, Stein SW, Leevy CM, Davidson CS. Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. J Clin Invest. 1960;39(4):592–600. doi:10.1172/JCI104072
- 17.↑
Suh YJ, Choi JY, Chai YJ, et al. Indocyanine green as a near-infrared fluorescent agent for identifying parathyroid glands during thyroid surgery in dogs. Surg Endosc. 2014;29(9):2811–2817. doi:10.1007/s00464-014-3971-2
- 18.↑
Bates D, Maechler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw. 2015;67(1):1–48. doi:10.18637/jss.v067.i01
- 19.↑
Ordinal-regression models for ordinal data. Version 2023.12-4.1. Accessed May 10, 2024. https://cran.r-project.org/web/packages/ordinal/index.html
- 20.↑
Sound S, Okoh A, Yigitbas H, Yazici P, Berber E. Utility of indocyanine green fluorescence imaging for intraoperative localization in reoperative parathyroid surgery. Surg Innov. 2015;26(6):774–779. doi:10.1177/1553350615613450
- 21.
Yu HW, Chung JW, Yi JW, et al. Intraoperative localization of the parathyroid glands with indocyanine green and firefly(R) technology during BABA robotic thyroidectomy. Surg Endosc. 2017;31(7):3020–3027. doi:10.1007/s00464-016-5330-y
- 22.
Cui L, Gao Y, Yu H, et al. Intraoperative parathyroid localization with near-infrared fluorescence imaging using indocyanine green during total parathyroidectomy for secondary hyperparathyroidism. Sci Rep. 2017;7(1):8193. doi:10.1038/s41598-017-08347-6
- 23.↑
Llorente PM, Barrasa AG, Martinez JMF, Prats MA, Sole MP. Intraoperative indocyanine green angiography of parathyroid glands and the prevention of post-thyroidectomy hypocalcemia. World J Surg. 2022;46(1):121–127.
- 24.↑
Priyanka S, Sam ST, Rebekah G, et al. The utility of indocyanine green (ICG) for the identification and assessment of viability of the parathyroid glands during thyroidectomy. Updates Surg. 2022;74(1):97–105. doi:10.1007/s13304-021-01202-4
- 25.↑
Van den Bos J, Wieringa FP, Bouvy ND, Stassen LPS. Optimizing the image of fluorescence cholangiography using ICG: a systematic review and ex vivo experiments. Surg Endosc. 2018;32(12):4820–4832. doi:10.1007/s00464-018-6233-x
- 26.
Larose PC, Brisson BA, Sanchez A, Monteith G, Singh A, Zhang M. Near-infrared fluorescence cholangiography in dogs: a pilot study. Vet Surg. 2024;53(4):659–670. doi:10.1111/vsu.14007
- 27.↑
Kono Y, Ishizawa T, Tani K, et al. Techniques of fluorescence cholangiography during laparoscopic cholecystectomy for better delineation of the bile duct anatomy. Medicine (Baltimore). 2015;94(25):e1005. doi:10.1097/MD.0000000000001005
- 28.↑
Eiger SN, Bertran J, Reynolds PS, et al. Use of near-infrared fluorescence angiography with indocyanine green to evaluate direct cutaneous arteries used for canine axial pattern flaps. Vet Surg. 2024;53(6):1073–1082. doi:10.1111/vsu.14121
- 29.↑
Aoki T, Yasuda D, Shimizu Y, et al. Image-guided liver mapping using fluorescence navigation system with indocyanine green for anatomical hepatic resection. World J Surg. 2008;32(8):1763–1767. doi:10.1007/s00268-008-9620-y
